Potential of Ginkgo biloba L. leaves in the management of hyperglycemia and hypertension using in vitro models

Leaves from four different Ginkgo biloba L. trees (1 and 2 – females; 3 and 4 – males), grown at the same conditions, were collected during a period of 5 months (from June to October, 2007). Water and 12% ethanol extracts were analyzed for total phenolics content, antioxidant activity, phenolic profile, and the potential in vitro inhibitory effects on α-amylase, α-glucosidase, and Angiotensin I-Converting Enzyme (ACE) enzymes related to the management of diabetes and hypertension. The results indicated a significant difference among the trees in all functional benefits evaluated in the leaf extracts and also found important seasonal variation related to the same functional parameters. In general, the aqueous extracts had higher total phenolic content than the ethanolic extracts. Also, no correlation was found between total phenolics and antioxidant activity. In relation to the ACE inhibition, only ethanolic extracts had inhibitory activity.     

A homozygous mutation in LTBP2 causes isolated microspherophakia

Microspherophakia is an autosomal-recessive congenital disorder characterized by small spherical lens. It may be isolated or occur as part of a hereditary systemic disorder, such as Marfan syndrome, autosomal dominant and recessive forms of Weill-Marchesani syndrome, autosomal dominant glaucoma–lens ectopia–microspherophakia–stiffness–shortness syndrome, autosomal dominant microspherophakia with hernia, and microspherophakia-metaphyseal dysplasia. The purpose of this study was to map and identify the gene for isolated microspherophakia in two consanguineous Indian families. Using a whole-genome linkage scan in one family, we identified a likely locus for microspherophakia (MSP1) on chromosome 14q24.1–q32.12 between markers D14S588 and D14S1050 in a physical distance of 22.76 Mb. The maximum multi-point lod score was 2.91 between markers D14S1020 and D14S606. The MSP1 candidate region harbors 110 reference genes. DNA sequence analysis of one of the genes, LTBP2, detected a homozygous duplication (insertion) mutation, c.5446dupC, in the last exon (exon 36) in affected family members. This homozygous mutation is predicted to elongate the LTBP2 protein by replacing the last 6 amino acids with 27 novel amino acids. Microspherophakia in the second family did not map to this locus, suggesting genetic heterogeneity. The present study suggests a role for LTBP2 in the structural stability of ciliary zonules, and growth and development of lens.     

The window and mechanisms of major age-related decline in the production of new neurons within the dentate gyrus of the hippocampus

While it is well known that production of new neurons from neural stem/progenitor cells (NSC) in the dentate gyrus (DG) diminishes greatly by middle age, the phases and mechanisms of major age-related decline in DG neurogenesis are largely unknown. To address these issues, we first assessed DG neurogenesis in multiple age groups of Fischer 344 rats via quantification of doublecortin-immunopositive (DCX+) neurons and then measured the production, neuronal differentiation and initial survival of new cells in the subgranular zone (SGZ) of 4-, 12- and 24-month-old rats using four injections (one every sixth hour) of 5'-bromodeoxyuridine (BrdU), and BrdU-DCX dual immunostaining. Furthermore, we quantified the numbers of proliferating cells in the SGZ of these rats using Ki67 immunostaining. Numbers of DCX+ neurons were stable at 4-7.5 months of age but decreased progressively at 7.5-9 months (41% decline), 9-10.5 months (39% decline), and 10.5-12 months (34% decline) of age. Analyses of BrdU(+) cells at 6 h after the last BrdU injection revealed a 71-78% decline in the production of new cells per day between 4-month-old rats and 12- or 24-month-old rats. Numbers of proliferating Ki67+ cells (putative NSCs) in the SGZ also exhibited similar (72-85%) decline during this period. However, the extent of both neuronal differentiation (75-81%) and initial 12-day survival (67-74%) of newly born cells was similar in all age groups. Additional analyses of dendritic growth of 12-day-old neurons revealed that newly born neurons in the aging DG exhibit diminished dendritic growth compared with their age-matched counterparts in the young DG. Thus, major decreases in DG neurogenesis occur at 7.5-12 months of age in Fischer 344 rats. Decreased production of new cells due to proliferation of far fewer NSCs in the SGZ mainly underlies this decline.     

An approach to obtain specific polyclonal antisera to Xanthomonas campestris pv. cyamopsidis and its potential application in indexing of infected seeds of guar

Clusterbean seed health testing is warranted since the pathogen (Xanthomonas campestris pv. cyamopsidis (Xccy)) is seed-borne and seed-transmitted. A polyclonal antibody was developed in rabbit via subcutaneous and intramuscular injections and characterized for sensitivity, specificity and its applicability to ELISA which: (i) was sensitive in detecting as few as 102 cells ml - 1 at a titre of 1: 4000; (ii) was specific, since it reacted only with Xccy and not with other xanthomonads; (iii) reacted both with Xccy cells and culture filtrate, indicating that the antigenic determinant is a secretory component; (iv) was applicable and reliable in seed health testing since it reacted only with infected seeds and plant materials and not with healthy seeds and (v) a purified fraction of antibody was virulent-specific since heat-denatured and avirulent isolates were not detected. The ELISA thus developed is highly reproducible and therefore suitable for the evaluation of the potential disease status of seeds and plant health, which is appropriate for routine seed health testing.     

CyP40, but not Hsp70, in rabbit reticulocyte lysate causes the aryl hydrocarbon receptor-DNA complex formation

Upon ligand binding, the aryl hydrocarbon receptor (AhR) translocates into the nucleus and dimerizes with its partner aryl hydrocarbon receptor nuclear translocator (Arnt). The AhR-Arnt heterodimer binds to the dioxin response element (DRE) to regulate target gene expression. Using baculovirus expressed human AhR and Arnt, we showed that the formation of the ligand-dependent AhR-Arnt-DRE complex requires protein factors in vitro. Recently, we provided evidence that p23, an Hsp90-associated protein, is involved in the complex formation. The aim of this study was to determine whether two other Hsp90-associated proteins present in rabbit reticulocyte lysate (RRL), namely CyP40 and Hsp70, play any role in forming the AhR-Arnt-DRE complex. Fractionation and immunodepletion experiments revealed that Hsp70 is not necessary for the formation of this complex. In contrast, CyP40 is involved in forming the complex since (1) immunodepletion of CyP40 from a RRL fraction reduces the intensity of the AhR-Arnt-DRE complex by 48% and (2) recombinant human CyP40 alone causes the formation of this complex. In addition, CyP40-interacting proteins appear to be essential for the full CyP40 effect on the AhR gel shift complex.     

Environmental surveillance system to track wild poliovirus transmission

Eradication of poliomyelitis from large metropolis cities in India has been difficult due to high population density and the presence of large urban slums. Three paralytic poliomyelitis cases were reported in Mumbai, India, in 1999 and 2000 in spite of high immunization coverage and good-quality supplementary immunization activities. We therefore established a systematic environmental surveillance study by weekly screening of sewage samples from three high-risk slum areas to detect the silent transmission of wild poliovirus. In 2001, from among the 137 sewage samples tested, wild poliovirus type 1 was isolated from 35 and wild poliovirus type 3 was isolated from 1. Acute flaccid paralysis (AFP) surveillance indicated one case of paralytic poliomyelitis from the city. Phylogenetic analysis with complete VP1 sequences revealed that the isolates from environmental samples belonged to four lineages of wild polioviruses recently isolated from poliomyelitis cases in Uttar Pradesh and not to those previously isolated from AFP cases in Mumbai. Wild poliovirus thus introduced caused one case of paralytic poliomyelitis. The virus was detected in environmental samples 3 months before. It was found that wild polioviruses introduced several times during the year circulated in Mumbai for a limited period before being eliminated. Environmental surveillance was found to be sensitive for the detection of wild poliovirus silent transmission. Nucleotide sequence analysis helped identify wild poliovirus reservoir areas.     

Advantages of fine-grained side chain conformer libraries

We compare the modelling accuracy of two common rotamer libraries, the Dunbrack-Cohen and the 'Penultimate' rotamer libraries, with that of a novel library of discrete side chain conformations extracted from the Protein Data Bank. These side chain conformer libraries are extracted automatically from high-quality protein structures using stringent filters and maintain crystallographic bond lengths and angles. This contrasts with traditional rotamer libraries defined in terms of chi angles under the assumption of idealized covalent geometry. We demonstrate that side chain modelling onto native and near-native main chain conformations is significantly more successful with the conformer libraries than with the rotamer libraries when solely considering excluded-volume interactions. The rotamer libraries are inadequate to model side chains without atomic clashes on over 20% of targets if the backbone is held fixed in the native conformation. An algorithm is described for simultaneously modelling both main chain and side chain atoms during discrete ab initio sampling. The resulting models have equivalent root mean square deviations from the experimentally determined protein loops as models from backbone-only ensembles, indicating that all-atom modelling does not detract from the accuracy of conformational sampling.     

SAMP14, a novel,acrosomal membrane-associated,glycosylphosphatidylinositol-anchored member of the Ly-6/urokinase-type plasminogen activator receptor superfamily with a role in sperm-egg interaction

We report a new member of the Ly-6/urokinase-type plasminogen activator receptor (uPAR) superfamily of receptors, SAMP14, which is retained on the inner acrosomal membrane of the human spermatozoan following the acrosome reaction and may play a role in fertilization. The SAMP14 sequence predicted a glycosylphosphatidylinositol (GPI)-anchored protein with a signal peptide, a transmembrane domain near the carboxyl terminus, and a putative transamidase cleavage site in the proprotein. Attachment of SAMP14 to the membrane by a lipid anchor was confirmed by its sensitivity to phosphatidylinositol phospholipase C. SAMP14 has a single functional domain similar to the Ly-6 and urokinase plasminogen activator receptor superfamily of proteins, and the gene mapped to 19q13.33, near the PLAUR locus for uPAR at 19q13.2. Northern and dot blotting showed that SAMP14 expression was testis-specific. Indirect immunofluorescence and immunoelectron microscopy with antisera to purified recombinant SAMP14 localized the protein to outer and inner acrosomal membranes as well as the acrosomal matrix of ejaculated human sperm. Acrosome-reacted sperm demonstrated SAMP14 immunofluorescence, indicating its retention on the inner acrosomal membrane following the acrosome reaction. However, SAMP14 localized to the entire sperm when unwashed swim-up sperm from the ejaculate were stained, indicating that some SAMP14 is loosely associated with the plasma membrane. Antibodies against recombinant SAMP14 inhibited both the binding and the fusion of human sperm to zona free hamster eggs, suggesting that SAMP14 may have a role in sperm-egg interaction. SAMP14 represents a GPI-anchored putative receptor in the Ly-6/uPAR family that is exposed on the inner acrosomal membrane after the acrosome reaction.     

RPE65 gene: multiplex PCR and mutation screening in patients from India with retinal degenerative diseases

We used multiplex PCR followed by sequencing to screen for mutations in the 14 exons of theRPE65 gene in early-hildhood-onset autosomal recessive retinitis pigmentosa (arRP) and Leber’s congenital amaurosis (LCA) patients. The RPE65 protein is believed to play an important role in the metabolism of vitamin A in the visual cycle and mutations identified in the gene could have implications for vitamin A-based therapeutic intervention. We were able to identify a homozygous mutation (AAT → AAG) in exon 9 in an arRP patient and a heterozygous missense transversion (AAT → AAG) also in exon 9 of an LCA patient. We also identified a polymorphism in exon 10 (GAG → GAA) in an arRP as well as an LCA patient. Mutation screening would be greatly facilitated by multiplex PCR which could cut down costs, labour and time involved. The nucleotide changes observed in this study could bede novo. Though a larger study has been undertaken, from the preliminary results it appears that in India theRPE65 gene seems to be less involved in causation of LCA.     

Development of ELISA for the detection of Ralstonia solanacearum in tomato: its application in seed health testing

Bacterial wilt caused by Ralstonia (formerly Pseudomonas) solanacearum is worldwide in distribution. It is one of the most destructive bacterial diseases of economically important crops. The serological assays so far developed for the detection of R. solanacearum were able to provide information as to the presence or absence of the pathogen in soil and plant materials. However, they could not discriminate between virulent and avirulent strains of the pathogen and were not specific to strains and races. In the present investigation, virulent bacterial cells (encapsulated with mucin) from tomato seeds were used as antigen and polyclonal antisera were developed in rabbit using a classical immunization protocol. Antisera thus developed were examined for the antibody titre, sensitivity, specificity, rapidity and the efficacy of the antibody in identifying the virulent and avirulent strains of the pathogen and the potential for application of this assay to the screening of infected plant materials and seeds. Our results indicate that the anti-tomato R. solanacearum: (i) has a good antibody titre of 1:10,000; (ii) can detect as few as 100 bacterial cells/ml; (iii) is tomato-specific (it reacted with tomato R. solanacearum, and not with isolates from chilli or eggplant); (iv) is reactive to all isolates of R. solanacearum from tomato; (v) is not cross-reactive with non-pseudomonads; (vi) is virulent strain-specific as it recognizes the virulent exopolysaccharide component as an antigenic determinant; (vii) reactivity could be correlated well with the degree of infection in tomato seeds and plant materials. The enzyme linked immunosorbent assay developed is sensitive, specific and rapid, therefore suitable for the detection of R. solanacearum isolates from tomato seeds during routine assays.